1. Field of the Invention
The present invention enters the medical field, for diagnosing and following up the time-dependent change in tissue fibrosis.
For this purpose, the invention particularly relates to a system for detecting and quantifying tissue fibrosis.
2. Description of Related Art Including Information Disclosed Under 37 CFR 1.97 and 37 CFR 1.98.
Tissue fibrosis refers to the accumulation of extracellular matrix within the connective tissue. Fibrosis occurs following an inflammation or a lesion of said tissue. The latter will not naturally and properly regenerate.
Fibrosis is a process for accumulating collagen which is synthesized by cells in the connective tissue. Collagen is a protein family physiologically present in the body notably for giving the tissue its mechanical resistance properties upon stretching. However, within the parenchyma of an organ, the excessive accumulation of collagen, therefore of resulting connective tissue of the fibrosis, causes a gradual, often inexorable, of the function of the organ.
Therefore, there exist several types of fibroses altering the function of the organs and causing diverse pathologies.
One refers to lung fibrosis in order to designate alteration of the lung connective tissue, the alveola of which are then found clasped and smothered by collagen fibers.
One refers to liver fibrosis for a fibrous connective tissue developing where liver cells have been destroyed. When liver fibrosis extends, it may result in the development of cirrhosis.
One also refers to kidney fibrosis which corresponds to an excessive accumulation of extracellular matrix in the renal parenchyma.
In other words, there exists a close correlation between the degree of fibrosis of an organ and its function. Generally, the more excessive is the accumulation of collagen in connective tissues, the larger is the loss of the function of the organ.
Accordingly, specifically quantifying tissue fibrosis is necessary in order to define a vital or functional prognosis and for following up the time-dependent change in an existing fibrosis.
In a known way, quantification of the fibrosis of a tissue is achieved on colored slides. An anatomopathologist will establish a score depending on the relative surface area occupied by fibrosis within the biopsy.
By “biopsy” is meant the fact of sampling a quite small portion of an organ or of a tissue in order to carry out medical examinations. This analysis technique on colored slides has the disadvantage of being subjective since it is not achieved by the human eye. This means for quantifying fibrosis therefore has poor intra-user and inter-user reproducibility, and therefore this quantification is not very representative and lacks reliability.
In order to overcome this limitation of the subjective quantification on colored slides, producing a semi-automated colorimetric quantification is also known. This technique consists in a semi-automated and computerized analysis based on automatic segmentation of the colors present on the slides colored with Masson Trichromium. The technique consists of setting tissue biopsies, dehydrating them, and then including them in paraffin. Sections of paraffin of four to ten micrometers (μm) are spread out on supports of the slide type, notably compatible with Fourier Transform Infrared Spectroscopy (“FTIRS”) and then colored with Masson Trichromium. Masson Trichromium colors in green the collagen fibers.
After staining with Masson Trichromium, the slides are scanned and then, from the obtained images, an operator selects the confusion areas appearing in green, i.e. the areas where collagen is physiologically present. The image after selection then undergoes a computerized processing based on an algorithm based on the segmentation of colors, identifying over three channels the red, green and blue colors, the green corresponding to the connective tissue with an excess of collagen and therefore to the fibrosis.
However, this semi-automated colorimetric quantification has the drawback of requiring a manual step for removing the confusion areas, introducing a share of subjectivity and an additional processing time.
Further, to this day, this technique has only been validated at renal tissue. Moreover, this technique systematically over-evaluates renal fibrosis. Indeed, the renal tubular basal membranes are made up of collagen, therefore colored in green and considered as fibrosis. This drawback is all the more important since the tubes are jointed and therefore the interstitial fibrosis is minimal. Thus, on a biopsy without any fibrosis, a 15 to 20% quantification of fibrosis is obtained, displaying an over-estimation of said quantification.
The semi-automatic colorimetric quantification does not allow differentiation of the different types of collagens within a same tissue, since it is based on a green coloration coloring all the collagens. Although reproducible for renal tissue fibrosis, the semi-automated colorimetric quantification therefore lacks representativity.
An equivalent technique uses Raman spectroscopy. It is also known for quantifying fibrosis, notably at the hepatic level, the Second-Harmonic Generation technique (SHG) which allows achieving a semi-automated quantification of collagen of type I present in the tissues. The SHG technique is based on the specific emission of collagen of type I after excitation by a two-photon microscope. After setting the tissue biopsies, dehydrating and inclusion in paraffin, sections from 4 to 6 μm were made and then spread out on supports compatible with FTIR, without any coloration. The tissue biopsies present on the slides are segmented into several fragments and then concatenated by the system. After excitation, the collagen of type I will emit an SHG signal. On each section, the SHG signal is detected. The tissue SHG signal is quantified via a semi-automated digital processing operation. An operator will have to define for each image or group of images the noise and positivity threshold values of the SHG signal. A quantification algorithm thus processes each image. The algorithm will calculate as a result the relative surface area occupied by the SHG signal, specific to the collagen of type I, for each biopsy according to the thresholds defined by the operator. The result of this is that this technique has as a drawback an intervention of the user who has to subjectively determine a positivity threshold of the SHG signal.
Further, the SHG imaging has been described exclusively for liver and lung fibrosis.
Further, the SHG technique has as another drawback of not distinguishing the fibrosis of the histological structures including collagen of type I physiologically like normal connective tissues. This SHG technique then allows detection of the whole of the collagen of type I present in the tissue.
The object of the present invention is to overcome the drawbacks of the methods mentioned earlier, by proposing a system for detecting fibrosis which is capable of quantifying and of following the time-dependent change of tissue fibrosis in an automatic reproducible and objective way by doing without the intervention of the operator. This system allows distinction of the different collagen histological structures, i.e. the connective tissue involved in the pathological tissue fibrosis of the connective tissue physiologically present.